Earticle

We isolated human mesenchymal stem cells (hMSC) from dental pulp. These cells are multipotent and serve as precursors for various mesoderm-type cells such as osteoblasts, chondroblasts and adipocytes, as well as several other types of cells. This study investigated the possible role of gangliosides in osteoblast differentiation of hMSCs. Gangliosides, which exist in glycosphingolipid-enriched domains on the cell membrane The role of gangliosides that play in osteoblastogenesis is not yet clearly understood. Therefore, in this study, We investigated the relationship between gangliosides and osteoblast differentiation of human mesenchymal stem cells (hMSCs). The results of high-performance thin-layer chromatography (HPTLC) showed that ganglioside GD1a expression was increased during the differentiation of hMSCs into osteoblasts. Furthermore, we showed that involvement of alkaline phosphate (ALP) with GD1a by immunostaining. Taken together, these results suggest that gangliosides GD1a may play a role in the osteoblast differentiation process of hMSCs.

Ganglioside are ubiquitous membrane component in mammalian cells and suggested to play important roles in various cell functions such as cell-cell recognition, differentiation and transmembrane signaling. These compounds are localized in a glycosphingolipid-enriched microdomain on the cell surface and regulated by the glycosphingolipid composition. However, the role that gangliosides play in immune rejection response by xenotransplantation is not yet clearly understood. In this study, we differential expression patterens of gangliosides between HEK293 cells (human embryonic kidney cells) and mini-pig kidney cells (NIH-mini pig, primary cultured) was investigated. we examined expression level of high-performance thin-layer chromatography (HPTLC) showed that HEK293 cells and mini-pig kidney cells contained GM3, GM2 and GD3 as major gangliosides. Moreover, ganglioside GM3, GM2, GM1 and GD3 which are gangliosides of mini-pig kidney cells expressed more than HEK293 cells. Especially, ganglioside GT1b expressed in HEK293 cells, not in mini-pig kidney cells. and GM1 expressed in mini-pig kidney cells, not in HEK293 cells. As a results suggest that differential expression patterens of gangliosides of HEK293, mini-pig kidney cells are related immune rejection response in xenotransplantation by biomaker.

Recent studies demonstrated that human mesenchymal stem cells (hMSCs) share molecular and cellular characteristics with bone marrow-derived mesenchymal stem cells (MSCs). hMSCs have the potential to differentiate into neural cells. Gangliosides are complex glycophingolipids with one or more sialic acids, the major components of cytoplasmic cell membranes. Numerous studies confirmed that the types of gangliosides and their expression levels are developmentally controlled and cell type-specific. Our recent studies proposed that expression of gangliosides is closely related to neural differentiation of embryonic stem cells in vitro. In this study, we investigated whether hMSCs are capable of differentiating into neural cells, and we analyzed the role of gangliosides in the neuronal differentiation of hMSCs. FACs analysis showed that the established cells have MSC characteristics. The cells did expressed MSC-specific surface antigene (CD44 and CD105), but not the hematopoietic markers CD45 and CD117. Next, hMSCs were differentiated into neuronal cell with induction media for 30h. Neuronal induced cells maintenance with differentiation media for 2weeks. High-performance thin-layer chromatography (HPTLC) showed that gangliosides GM3, GD3 and GD1a expressed in differentiated into neuronal cells for 2weeks, especially GD3 increased comparison to control cell. Immunofluorescence staining also agreed with the results of HPTLC analysis. These differentially expressed gangliosides suggest that gangliosides may have specific functions in stem cell and during neuronal differentiation. Therefore, thess results also suggest that regulation of gangliosides expression have used as differentiation marker of neuronal cell from hMSCs.

High-throughput quantitative analytical method for plant N-glycan has been developed. All steps, including peptide N-glycosidase (PNGase) A treatment, glycan preparation and exoglycosidase digestion, were optimized for high-throughput applications using 96-well format procedures and automatic analysis on a DNA sequencer. The glycans of horseradish peroxidase with plant-specific core α(1,3)-fucose can be distinguished by the comparison of the glycan profiles obtained via PNGase A and F treatments. The peaks of the glycans with (91%) and without (1.2%) α(1,3)-fucose could be readily quantified and shown to harbor bisecting β(1,2)-xylose via simultaneous treatment with α(1,3)-mannosidase and β(1,2)-xylosidase. This optimized method was successfully applied to analyze N-glycans of plant-expressed recombinant antibody, which was engineered to contain a minor amount of glycan harboring β(1,2)-xylose. These results indicate that our DNA sequencer-based method provides quantitative information for plant-specific N-glycan analysis in a high-throughput manner, which has not previously been achieved by glycan profiling based on mass spectrometry.

β-O-linkedN-acetylglucosamine(O-GlcNAc) is a nucleocytosolic post-translational modification on serine and threonine residues that is dynamically regulated by O-GlcNActransferase and O-GlcNAcase. Many proteins are O-GlcNAcylated in response to various cellular processes, including transcription, proliferation, apoptosis and signal transduction. So, we have studied the function of O-GlcNAc modification on several proteins. We found that O-GlcNAcylated NF-kB was translocated into nucleus and had increased transcriptional activity. We report increased O-GlcNAcylation in non-small cell lung carcinoma A549 cells and various other cells in response to glucose deprivation. Also we show that glycogen degradation in response to glucose deprivation provides a source for UDP-GlcNAc required for increased O-GlcNAcylation under this condition. we demonstrate that Snail is O-GlcNAcylated and adjacent-site occupancy inhibits phosphorylation by GSK-3, resulting in increased Snail stability and attenuation of E-cadherin proximal promoter activity and transcription level. Furthermore, Overexpression of OGT induces in vivo invasion program of epithelial cancer cells by Snail-dependent manner. O-GlcNAc modification seems to be involved in neurite outgrowth in cultured neuronal cells. O-GlcNAcylation is highly conserved, so we were able to find several novel proteins modified with O-GlcNAc in Drosophila S2 cell line. Also we found O-GlcNAcylated site in ncOGT outside of TPR by using Q-TOF MS. Now we focus on the activities and function of ncOGT by mutagenesis studies. As stated above, O-GlcNAcylation is related to many cellular metabolism. And we already have found the function of O-GlcNAc modification on several important proteins. We'll keep on concentrating on this interesting post-translational modification. If you have any interest, feel free to contact us.

It has been reported that one downstream effector produced from glucose is uridine diphosphate-N-acetly glucosamine (UDP-GlcNAc) via the hexoamine biosynthetic pathway (HBP). The dynamic cycle of addition and removal of O-linked-N-acetlyglucosamine (O-GlcNAc) to Ser/Thr residues is involved in regulating nuclear and cytoplasmic proteins. Nucleocytoplasmic O-linked GlcNAc transferases(ncOGT) which add a single GlcNAc to hydroxyl groups of serine and threonine residues. ncOGT is characterized by an amino terminuns bearing tetratricopeptide repeats (TPR), possible nuclear trafficking motifs, and a catalytic domain within its C-terminus. Interestingly, O-GlcNAc glycosylation occurs in nucelocytoplsmic O-linked GlcNAc transferase (ncOGT) as well and several sites have been identified mainly within TPR domain. We found O-GlcNAcylated site in ncOGT outside of TPR by using Q-TOF MS. Now we focus on the activities and function of ncOGT by mutagenesis studies. This ongoing effort would give us clear understanding of the key enzyme of O-GlcNAc metabolism and how the O-GlcNAc modification may be regulated.

The transcriptional repressor Snail plays a key role in epithelial-mesenchymal transition (EMT) by which direct repression of E-cadherin transcription. Therefore, regulation of Snail expression level in epithelial tumor cells is important not only for maintaining of epithelial homeostasis, but also for invasion and metastasis of cancer cells by the EMT program. Series of Ser imbedded in Snail are phosphorylated by GSK3 and Snail expression is dynamically regulated by Wnt signaling together with β-catenin while driving a Snail-dependent EMT program. Glucose flux through the hexosamine biosynthetic pathway (HBP) can be used for the source of O-linked β-N-acetylglucosamine(O-GlcNAc) modification on Ser and Thr residues of various nucleocytoplasmic proteins. In this study, we demonstrate that Ser112 of Snail is O-GlcNAcylated and this adjacent-site occupancy inhibits phosphorylation by GSK-3, resulting in increased Snail stability and attenuation of E-cadherin proximal promoter activity and transcription level. Furthermore, Overexpression of OGT induces in vivo invasion program of epithelial cancer cells by Snail-dependent manner. Taken together, our results indicate dynamic interplay between O-GlcNAcylation and GSK-3 phosphorylation of Snail, and our observations may provide the molecular insight of pathogenic and prognostic correlation between cancer progression and hyperglycemic condition of diabetes.

Isomaltooligosaccharide (IMO) is a promising dietary component with prebiotic effect, and the long-chain IMOs are preferred to short chain ones owing to the longer persistence in the colon. To establish the optimal process for synthesis of long-chain IMOs, we systematically examined the reaction condition of dextransucrase of Leuconostoc mesenteroides B-512F by changing the ratio of sucrose to maltose (varying as 1:4, 1:2, 1:1, and 2:1) and amount of each sugar (from 2% to 20%). As a result, a ratio of 2:1 (sucrose to maltose, 10:5% or 20:10%, w/v) was determined as an optimal condition for long-chain IMO synthesis (DP3-DP9) with relatively higher yields (70-90%, respectively).

Cancer is often difficult to early diagnose, but early diagnosis is a crucial factor for good outcome in the cancer. N-acetylglucosaminyltransferase V catalyzes an addition of b1,6-N-acetylglucosamine (GlcNAc) to the core N-glycan, many lines of evidence have demonstrated the role of N-acetylglucosaminyltransferase V (GnT-V) in the pathogenecity of colon cancer cell. One of the related example is an aberrant TIMP-1-mediated cancer progression in which deterioration of the quality of TIMP-1 is induced by glycosyl alterations through GnT-V catalysis, leading to mitigated MMP inhibition responsible for an strengthened invasive/metastatic potential of cancer cells. Accordingly the identification of target proteins for GnT-V has been an important subject for cancer biomarker discovery. We developed a protocol in which L-PHA, a lectin recognizing b1,6-GlcNAc, is conjugated to avidin-agarose complex to capture b1,6-GlcNAc-carrying serological glycoproteins and the captured glycoproteins were identified using an LTQ-FTICR mass spectrometer. Candidate proteins showing differential amounts between normal and cancer sera were confirmed by western blot analysis and will be subject to validation for valid biomarker for colon cancer.